Abstract Influenza virus is a major public health burden that is currently controlled by yearly vaccination. Protection is largely conferred by antibodies. However, public health officials fear that such immunizations might miss newly emerging viruses or those with novel subtypes of hemagglutinin or neuraminidase proteins, with the potential to lead to a pandemic. Moreover, the costs of the current approach represent a major burden to society. Recently, hope has arisen that it may be possible to formulate a universal vaccine, capable of conferring resistance to numerous subtypes. Rare monoclonal antibodies targeting the stem region of influenza virus can be broadly neutralizing, illustrating the possibility of raising a universal influenza virus vaccine, but also indicating that the natural response is limited or natural influenza antigens do not stimulate the immune system in an appropriate way. Broadly neutralizing human antibodies reactive to the stem of influenza usually use VH1-69 with moderate numbers of mutations, but have few other features in common. The VH1-6+D3-3 broadly neutralizing antibody class targets the stem in a distinct way and has been seen in independent patients. In this RO1 project, we propose to study existing and new knock- in mice carrying germline human VH1-69 or VH1-6+D3-3 targeted to the physiological loci for their responses to influenza vaccine candidates. We shall isolate new rationally designed immunogens by evolving HA variants or scaffolds with affinity for germline- reverted broadly neutralizing VH1-69 and VH1-6+D3-3 class antibodies. These novel immunogens will then be tested for the ability to stimulate B cells using germline VH1- 69 or VH1-6+D3-3 in vivo, using novel mouse strains generated for the purpose. Finally, we will assess the ability of these immunogens alone or together with boosting immunogens for the ability to confer broad neutralizing protection. Our long-term goals are to understand the limitations of the natural influenza antibody response, to learn how to optimize immunogens, and to identify candidate universal vaccine immunogens suitable for human trials.